Heterogeneity of polyclonal IgE characterized by differential charge, affinity to protein A, and antigenicity  Zhikang Peng, MDa,c, Gilbert Arthur, PhDb, Edward S. Rector, PhDc, Danuta Kierek-Jaszczuk, PhDc, F.R. Simons, MDa, Allan B. Becker, MDa,c  Journal of Allergy and Clinical Immunology  Volume 100, Issue 1, Pages 87-95 (July 1997) DOI: 10.1016/S0091-6749(97)70199-4 Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Chromatofocusing of dog IgE (pH 4.0 to 7.4). The dog IgE fraction was loaded on a polybuffer exchanger column and eluted from the column with polybuffer, pH 4 (described in Methods). A, Dog IgE (blank bar, measured by PCA), IgG, IgA, IgM (measured by ELISA), and protein. B, Dog IgE measured by PCA with different calculations. Blank bar shows PCA titer; dotted line shows skin blue area at dilution of 1:2; solid line shows blue area at dilution of 1:10. C, Dog IgE measured by PCA (titer) and ELISA with polyclonal and mAb C2 anti-dog IgE antibodies. IgE1 (eluted at pH 5.0) reacted with only polyclonal anti-dog IgE antibodies, whereas IgE2 (eluted at pH 4.7) reacted with both polyclonal and mAb C2 antibodies. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Chromatofocusing of dog IgE (pH 4.0 to 7.4). The dog IgE fraction was loaded on a polybuffer exchanger column and eluted from the column with polybuffer, pH 4 (described in Methods). A, Dog IgE (blank bar, measured by PCA), IgG, IgA, IgM (measured by ELISA), and protein. B, Dog IgE measured by PCA with different calculations. Blank bar shows PCA titer; dotted line shows skin blue area at dilution of 1:2; solid line shows blue area at dilution of 1:10. C, Dog IgE measured by PCA (titer) and ELISA with polyclonal and mAb C2 anti-dog IgE antibodies. IgE1 (eluted at pH 5.0) reacted with only polyclonal anti-dog IgE antibodies, whereas IgE2 (eluted at pH 4.7) reacted with both polyclonal and mAb C2 antibodies. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Chromatofocusing of dog IgE (pH 4.0 to 7.4). The dog IgE fraction was loaded on a polybuffer exchanger column and eluted from the column with polybuffer, pH 4 (described in Methods). A, Dog IgE (blank bar, measured by PCA), IgG, IgA, IgM (measured by ELISA), and protein. B, Dog IgE measured by PCA with different calculations. Blank bar shows PCA titer; dotted line shows skin blue area at dilution of 1:2; solid line shows blue area at dilution of 1:10. C, Dog IgE measured by PCA (titer) and ELISA with polyclonal and mAb C2 anti-dog IgE antibodies. IgE1 (eluted at pH 5.0) reacted with only polyclonal anti-dog IgE antibodies, whereas IgE2 (eluted at pH 4.7) reacted with both polyclonal and mAb C2 antibodies. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 IEF immunoblot for dog IgE, IgE1, and IgE2 fractions. The dog IgE fraction before chromatofocusing (strips 2, 5, and 8), the IgE1 (lanes 3, 6, and 9) and IgE2 (lanes 4, 7, and 10) fractions obtained from chromatofocusing, and dog IgG (lane 1) were separated by IEF. Separated proteins were either stained with Coomassie Brilliant Blue (lanes 1 to 4) or transferred onto nitrocellulose, followed by immunoblotting with polyclonal (lanes 5 to 7) or mAb C2 (lanes 8 to 10) anti-dog IgE. Lanes 5, 6, and 7 show that IgE, IgE1, and IgE2 fractions reacted with rabbit anti-dog IgE. Lanes 8, 9, and 10 show that IgE2 contained in the IgE fraction (lane 8) and the IgE2 fraction (lane 10) reacted with mAb C2 anti-dog IgE, whereas IgE1 fraction (lane 9) did not react with the mAb. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 Binding properties of dog IgE to protein A. Dog IgE fraction was loaded on a Protein A-Sepharose 4B column and then washed and eluted as described in Methods. Dog IgE was measured by PCA and ELISA with polyclonal and mAb C2 anti-dog IgE antibodies. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 4 Estimation of molecular mass for dog IgE, IgE1, and IgE2. Dog IgE (lane 1), IgE1 (lane 2), and IgE2 (lane 3) were separated by 5% to 15% gel SDS-PAGE under nonreducing conditions and electrotransferred onto nitrocellulose membranes. Membranes were incubated with rabbit anti-dog IgE, followed by incubation with enzyme-conjugated goat anti-rabbit IgG. Molecular mass of dog IgE was 226 kd, as indicated. Ab, Antibody. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 5 SDS-PAGE and immunoblot analysis of cross-reactivity of dog IgE1 and IgE2 to human and mouse IgE. Proteins of dog IgE-enriched fraction (lanes 1 and 4) and dog IgE1 (lanes 2 and 5) and IgE2 (lanes 3 and 6) fractions, as indicated at bottoms of lanes, were separated by SDS-PAGE on 10% gel and then transferred to nitrocellulose membranes. Membranes were immunoblotted with anti-human IgE (A) or anti-mouse IgE (B), followed by incubation with relevant conjugated anti-IgG antibodies. Lanes 1, 2, and 3 were incubated with polyclonal anti-IgE; and lanes 4, 5, and 6 were incubated with mAb anti-IgE. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 5 SDS-PAGE and immunoblot analysis of cross-reactivity of dog IgE1 and IgE2 to human and mouse IgE. Proteins of dog IgE-enriched fraction (lanes 1 and 4) and dog IgE1 (lanes 2 and 5) and IgE2 (lanes 3 and 6) fractions, as indicated at bottoms of lanes, were separated by SDS-PAGE on 10% gel and then transferred to nitrocellulose membranes. Membranes were immunoblotted with anti-human IgE (A) or anti-mouse IgE (B), followed by incubation with relevant conjugated anti-IgG antibodies. Lanes 1, 2, and 3 were incubated with polyclonal anti-IgE; and lanes 4, 5, and 6 were incubated with mAb anti-IgE. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 6 IEF immunoblot analysis of neuraminidase- and endoglycosidase-treated IgE1 and IgE2 fractions. Dog IgE1 (lanes 1, 2, 5, and 6) and IgE2 (lanes 3, 4, 7, and 8) obtained by chromatography on Protein A-Sepharose were incubated with indicated enzymes or relevant buffers as controls. A, Lanes 2, 4, 6, and 8 were incubated with neuraminidase; lanes 1, 3, 5, and 7 were incubated with buffer. B, Lanes 2, 4, 6, 8 were incubated with endoglycosidase F/N-Glycosidase F; lanes 1, 3, 5, and 7 were incubated with buffer. These incubations were then subjected to IEF immunoblot with mAb D9 (lanes 1 to 4) or mAb C2 anti-dog IgE (lanes 5 to 6), as described in Methods. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 6 IEF immunoblot analysis of neuraminidase- and endoglycosidase-treated IgE1 and IgE2 fractions. Dog IgE1 (lanes 1, 2, 5, and 6) and IgE2 (lanes 3, 4, 7, and 8) obtained by chromatography on Protein A-Sepharose were incubated with indicated enzymes or relevant buffers as controls. A, Lanes 2, 4, 6, and 8 were incubated with neuraminidase; lanes 1, 3, 5, and 7 were incubated with buffer. B, Lanes 2, 4, 6, 8 were incubated with endoglycosidase F/N-Glycosidase F; lanes 1, 3, 5, and 7 were incubated with buffer. These incubations were then subjected to IEF immunoblot with mAb D9 (lanes 1 to 4) or mAb C2 anti-dog IgE (lanes 5 to 6), as described in Methods. Journal of Allergy and Clinical Immunology 1997 100, 87-95DOI: (10.1016/S0091-6749(97)70199-4) Copyright © 1997 Mosby, Inc. Terms and Conditions